ESBL/pAmpC-Producing Escherichia coli Causing Urinary Tract Infections in Non-Related Companion Animals and Humans

Urinary tract infections (UTI) caused by Escherichia coli are frequently diagnosed in humans and companion animals. Extended-spectrum beta-lactamase (ESBL)- and cephalosporinase (pAmpC)-producing Escherichia coli are worldwide-disseminated and frequently multidrug-resistant, hence leading to treatment failure and public health concerns. This study aimed to characterize and compare ESBL/pAmpC-producing E. coli strains causing community-acquired UTI in companion animals and non-related humans. Third-generation cephalosporin (3GC)-resistant E. coli (companion animals n = 35; humans n = 85) isolated from patients with UTI were tested against 14 antimicrobials following CLSI guidelines. PCR-based assays were used to detect the major E. coli phylogenetic groups, pathogenicity associated-islands (PAIs), virulence genes, and ESBLs/pAmpC resistance genes. ESBL/pAmpC-producing E. coli isolates were typed by multi-locus sequence typing (MLST) and PCR. E. coli strains from companion animals and humans shared two MDR high-risk clonal lineages: ST131 and ST648. To the best of our knowledge, this study reports the first description of E. coli ST131 clade C1-M27 and the clonal lineage ST131 clade A in humans with community-acquired UTI in Portugal. Considering that companion animals with UTI are generally treated at home by the owners, measures should be implemented to avoid the spread of multidrug-resistant high-risk clones to humans and their household environment.


Introduction
Urinary tract infections (UTI) may be caused by the uropathogenic Escherichia coli (UPEC), which is one of the extraintestinal pathogenic E. coli pathotypes (ExPEC), and one of the most frequent etiologic agents of UTI worldwide both in humans and companion animals [1][2][3][4].
pAmpC-producing E. coli strains belonged mainly to group-D in both groups, and were significantly more frequent in companion animals (94.1% in E. coli from companion animal; 40.0% in E. coli from humans, p < 0.0001) ( Table 3).
Although bla SHV was detected among ESBL-producing E. coli, bla CTX-M-type ESBL clearly predominated in both groups. Several types of CTX-M enzymes were found showing high diversity of these ESBLs in UPEC, especially in strains from humans. Nevertheless, bla CTX-M-15 predominated in both groups (Table 4).   Regarding pAmpC, only bla CMY-2 was found, being the predominant antimicrobial resistance mechanisms responsible for 3GC-resistance in E. coli from companion animals. Interestingly, the cefoxitin resistance phenotype of four 3GC-resistant E. coli strains from companion animals could not be explained by any of the tested genes; thus, other mechanism of resistance were likely involved.
Moreover, carbapenemase genes were not detected in either 3GC-resistant E. coli collections, which is in line with the carbapenem-susceptible phenotype of these strains.
All E. coli strains were positive for the ecpA gene, the major pilin subunit of E. coli common pilus, in both groups. Furthermore, the papEF operon segment, iucD, hlyA, and cnf1 were also frequent in both groups (Table 6). However, the cytotoxic necrotizing factor-1 (cnf1 gene) and aerobactin siderophore (iucD gene) frequencies were significantly higher in E. coli strains from humans (p = 0.012 and p = 0.0002, respectively) ( Table 6).
Notably, ESBL/pAmpC-producing E. coli from companion animals and humans with UTI belonged to two MDR high-risk clonal lineages, namely the ST131 and ST648. Moreover, the ST88 and ST354 clonal lineages were also shared by companion animals and humans (Figures 7, S1 and S2).

Discussion
This study showed that ESBL/pAmpC-producing E. coli from companion animals and humans with UTI may harbor a big diversity of clinically relevant beta-lactamases and were associated with several virulence determinants.
E. coli strains isolated from companion animals were frequently associated with the presence of bla CTX-M-15 and bla CMY-2 genes, while those isolated from humans were associated with bla CTX-M-15 and bla CTX-M-1 . The high prevalence and disseminations of bla CTX-M-15 in E. coli isolated from animals and humans agrees with studies conducted worldwide [10]. Moreover, CMY-2-producing E. coli strains belonged mainly to the phylogenetic group-D, which is consistent with a previous study conducted in the United States [20].
Despite the similarities between the E. coli strains isolated from both study groups with respect to mobile genetic determinants of antimicrobial resistance and virulence, the phylogenetic group-B2 and group-D was significantly more common in humans and companion animals, respectively. This finding may point to different E. coli host species adaptations contributing both to the global dissemination of overlapping antimicrobial resistance and virulence determinants.
The E. coli phylogenetic group-A, which is usually considered commensal and less pathogenic, was found to cause UTI in both companion animals and humans. Furthermore, this phylogroup was associated with a high diversity of globally disseminated bla CTX-M genes, such as bla CTX-M-15 and bla CTX-M-32 . These findings highlight the high dissemination efficiency of plasmid-mediated beta-lactamases that may lead to therapeutic failure even in infections caused by less pathogenic strains.
Overall, 3GC-resistant E. coli strains belonging to the phylogenetic group-B2 had the higher number of PAI markers. This association of the group-B2 with several PAI markers is in line with previous reports of UPEC strains [21]. Interestingly, the most frequent PAI combination pattern was related to strains containing PAI IV536 and PAI ICFT073 . These PAI markers contain fimbrial adhesins and iron-uptake-system encoding genes that seem to be important for UPEC fitness and effective host colonization of the urinary tract. A high prevalence of fimbrial adhesin-encoding genes (such as, papEF operon segment) has been described in E. coli isolated from human patients diagnosed with UTI, thus highlighting the importance of these structures in the pathogenesis of UTI [22]. Furthermore, PAI ICFT073 also carries the toxin hemolysin A (hlyA gene), that is responsible for the creation of pores in the host cell membranes leading to cell lysis [22]. Notably, PAI IV536 and PAI ICFT073 were detected in 74.3% and 54.3% of E. coli strains from companion animals, respectively. Strains isolated from humans had an even higher prevalence of these PAIs (91.8% and 78.8%, respectively).
The uropathogenic-specific protein gene (usp) was detected only in one E. coli ST131 strain, which was isolated from a dog diagnosed with UTI in 2015. This protein is a genotoxin active against mammalian cells that can induce characteristics of apoptosis and has been associated with E. coli isolates from pyelonephritis, prostatitis, and bacteremia of urinary tract origin. It has been proposed that the usp gene provides immunity to its producer and enhances infectivity of the urinary tract [23,24].
Regarding the E. coli population structure, four sequence types were detected in companion animals and humans-the ST131 and ST648 MDR high-risk clonal lineages, and the ST88 and ST354 clonal lineages. The fact that the E. coli strains included in this study, from companion animals and humans, were collected in different years and from nonrelated patients is considered a study limitation, since it could have limited the detection of additional E. coli STs that are able to cause UTI in both groups. It should be noted that the results from this study may differ from studies including samples from a more recent timeframe as consequence of natural evolution and dissemination of beta-lactamases and E. coli clonal lineages. Nevertheless, the retrospective nature of this study is important as it contributes to the global understanding of the ecology of this common pathogen. Furthermore, this study includes data about virulence, which is still seldomly studied in strains from companion animals.
The ST131 clonal lineage harboring bla CTX-M-15 or bla CTX-M-14 has been detected in E. coli strains isolated from companion animals in many other countries [17,19,25]. However, the E. coli O25b:H4-ST131 harboring the bla CMY-2 gene has been rarely described [26,27]. In Japan, between 2005 and 2010, the bla CTX-M-14 gene was found to be the most common, followed by bla CTX-M-15 and bla CTX-M-2 [25]. In the present study, bla CTX-M-14 was also only detected in E. coli strains isolated from humans. The bla CTX-M-27 gene, a single-nucleotide variant of bla CTX-M-14 , is being increasingly detected among E. coli strains isolated from humans and companion animals with UTI in the United States, Asia, and Europe [25,[28][29][30]. In this study, the bla CTX-M-27 gene was only detected in one strain of human origin. To the best of our knowledge, this is the first description of the ST131 C1/H30R1 E. coli subclade C1-M27 and bla CMY-2 -producing E. coli O25b:H4-ST131 in humans with community-acquired UTI from Portugal. Some reports have documented an increase in the number of the ST131 C1/H30R1 E. coli clade C1-M27 since the late 2000s. Isolates of this emerging clade have been reported in clinical samples from humans of Japan, France, Germany, Berlin, Geneva, Madrid, and Utrecht [25,[31][32][33]; and also in companion animals, birds and urban seagulls [30,34,35]. Moreover, in northern Portugal, the C1-M27 clade has been isolated from fecal samples of healthy humans [36]. It is noteworthy that the ST131 C1/H30R1 E. coli clade C1-M27 has been shown to have a higher dissemination rate than the O25b:H4-ST131-H30Rx [37,38]. This dissemination rate may be accelerated by the expression of advantageous virulence determinants directed to the intestinal tract colonization, which is of particular importance to patients receiving antimicrobials or frequently admitted to hospital settings [33]. Thus, human colonization by clade C1-M27 should be monitored to improve preventive measures against infection and colonization of companion animals.
Previous studies about clinical E. coli strains showed that the O16-H5-ST131 clonal lineage (clade A) is globally distributed [39]. In this study, an E. coli O16:H5-ST131 harboring bla CTX-9-like was detected, which, to the best of our knowledge, is the first description in a human with community-acquired UTI from Portugal. The lower frequency of the O16-B2-ST131 in this study when compared to other countries [39][40][41], may reflect geographical and temporal differences in its distribution. Furthermore, according to Matsumura et al. [40], the O16-B2-ST131 clonal lineage has a lower prevalence of resistance to fluoroquinolones and ceftriaxone than O25b-B2-ST131 isolates [39][40][41]. This may also explain the low detection of O16-B2-ST131 in this study since it was focused on 3GC-resistant E. coli. Considering its high frequency in humans, the development of rapid molecular detection methods to identify ST131, while waiting for culture and antimicrobial susceptibility, could aid in a more effective initial antibiotic therapy and the reduction of its dissemination [39].
The E. coli ST648 high-risk clonal lineage is pandemic and globally reported in healthy and diseased humans and companion animals worldwide [13,20,[42][43][44][45][46][47][48]. E. coli ST648 isolated from human infection may harbor several ESBL/pAmpC and carbapenemases, contributing to its dissemination [13,42,49]. As in ST131, the E. coli ST648 is frequently associated with CTX-M enzymes, namely CTX-M-15 [13]. The 3GC-resistant E. coli ST648 strains from this study, were all found to be CMY-2-producers. Furthermore, the CMY-2-producing ST648 clonal lineage was very common among isolates from companion animals, which is in line with previous studies [20,43]. Being a clonal lineage that is high-risk to humans, monitoring of ST648 in companion animals is of the upmost importance.
Interestingly, the E. coli ST88 and ST354 clonal lineages have been associated with poultry and broiler meat, suggesting that farm animals may be reservoirs of E. coli that are able to cause extraintestinal diseases in humans and companion animals [26,[50][51][52][53][54]. E. coli ST354 has also been isolated from clinical samples of companion animals from Australia, and has been suggested to have a propensity to persist and circulate in animalcare facilities [55]. To the best of our knowledge, this is the first report of a CMY-2-producer E. coli ST354 in companion animals with UTI in Europe.
E. coli ST10 and ST410, other two important pandemic clonal lineages, were found in humans with UTI from this study [11,56]. E. coli ST410 seems to be a successful ExPEC clonal lineage like ST131 [57,58]. E. coli ST410 was first described, in 2016, in China [59] and since then has been reported worldwide in humans, companion animals, wildlife, and the environment [58,[60][61][62][63]. Notably, in 2017, the ST410 was detected in companion animals with UTI from China [45]. Nevertheless, only a few studies have detected this clonal lineage in E. coli isolated from companion animals [45,62,63]. The E. coli ST10 clonal lineage has been reported in samples from animals (birds, swine, and sheep), in healthy human feces, and in humans with UTI. This clonal lineage is also usually associated with several 3GC-resistance genes such as bla CTX-M-14 and bla CTX-M-15 [64][65][66][67]. However, in the present study, the ST10 clone was associated with bla CTX−M−1 . Although not detected in this study, ST410 has been previously described in isolates from companion animals, again, highlighting their possible role in the dissemination of high-risk clonal lineages.
The results from this study have high clinical relevance since it is shown that 3GCresistant E. coli strains causing UTI in companion animals not only may belong to MDR high-risk clonal lineages, but are also likely to harbor critically important mobile genetic determinants associated with high pathogenicity or antimicrobial resistance.

Bacterial Isolates
Three hundred and thirty non-duplicate uropathogenic E. coli (UPEC) were isolated, between 1999 to 2015, from companion animals with UTI at the Laboratory of Antibiotic Resistance, the Faculty of Veterinary Medicine, University of Lisbon, Portugal. Furthermore, 85 non-duplicate 3GC-resistant E. coli isolates from humans with community-acquired UTI were obtained in 2013 from a diagnostic laboratory in the Lisbon area. 3GC-resistance was determined by antimicrobial susceptibility testing using cefotaxime or ceftazidime as surrogates, as described in Section 4.2.
Identification and confirmation of the isolate species was performed by a previously described PCR targeting the E. coli gadA gene [68].
Antimicrobial categories were used to characterize multidrug resistance as previously proposed by Magiorakos et al. [71].

Molecular Detection of Antimicrobial Resistance Genes
DNA extraction was conducted using a boiling method [72]. Antimicrobial resistance genes were investigated in resistant and intermediate resistant strains.
3GC-resistant E. coli were screened for bla CTX-M-type genes by PCR [73]. Positive isolates for bla CTX-M-type were further tested by PCR for bla CTX-M-group1 , bla CTX-M-group2 , and bla CTX-M-group9 [74] and positive amplicons were submitted to nucleotide sequencing. Cefoxitin-resistant E. coli isolates were tested using a multiplex-PCR with specific primers targeting plasmid-borne genes encoding AmpC β-lactamases (bla CIT , bla LAT , bla ACT , bla MIR , bla FOX , bla MOX , and bla DHA ), as previously described [75]. Positive samples for the group CIT were submitted to nucleotide sequencing after PCR amplification targeting the entire bla CMY-2 gene [76]. 3GC-resistant E. coli negative for bla CTX-M-type or AmpC genes were tested for the presence of bla TEM-type and bla SHV-type ESBL genes [72].
Strains were screened by PCR for the presence of common carbapenemase genes (bla IMP , bla OXA , bla VIM , bla NDM , and bla KPC ), as previously described [77].

Uropathogenic Escherichia coli Phylogenetic Typing, Pathogenicity Island Markers, and Virulence Genotyping
Phylogenetic typing was performed in all 3GC-resistant E. coli strains to determine the main phylogenetic groups (A, B1, B2, and D) according to the amplification of chuA gene, yjaA gene, and TspE4C2 fragment [78].
E. coli strains not belonging to the ST131 clonal lineage were typed by MLST. Briefly, the seven housekeeping genes of the E. coli MLST scheme (adk, fumC, gyrB, icd, mdh, purA, and recA) were amplified by PCR using the primers and amplification conditions previously described in https://enterobase.warwick.ac.uk/ (accessed on 10 February 2020) [83]. PCR products were purified using the NZYTech Gel Pure Kit (NZYTech-Genes and Enzymes, Lisbon, Portugal) and sequencing was performed by Stabvida (Caparica, Portugal). Sequence quality was confirmed using Ugene Unipro software (Unipro, Novosibirsk, Russia) and the respective alleles and sequence types were retrieved using the publicly available E. coli MLST database.

Statistical Analysis
The SAS statistical software package for Windows v. 9.4 (SAS Institute Inc., Cary, NC, USA) was used for statistical analysis. The Fisher's exact test was used for comparisons between groups (two by two analysis of contingency tables) with a p value of 0.05.

Conclusions
This study showed that 3GC-resistant E. coli from companion animals and humans with community-acquired UTI frequently belong to important pandemic high-risk clonal lineages and harbor clinically relevant antimicrobial resistance and virulence determinants that are easily disseminated. Considering the close contact between companion animals and humans in modern society, the dissemination of pandemic E. coli clones, such as ST131-C2/H30Rx (bla CTXM-15 ) and ST648, in patients with UTI requires the joint action of human and veterinary medicine. Although the degree of inter-species transmission and zoonotic/zooanthroponotic potential of such bacteria is complex to evaluate, the high frequency of common PAI markers and CTX-M enzymes reported in this study, highlights that the link between humans and companion animals goes beyond sharing specific E. coli clones. As appraised by the scientific community, a One Health approach is required to fully grasp the dissemination dynamics of such bacterial clones and/or their antimicrobial resistance and virulence mobile genetic determinants.